Development of a rapid, in‐situ analysis method using sheath‐flow probe electrospray ionisation‐mass spectrometry for the direct identification of cocaine metabolites in dried blood spots

Rationale Small amounts of biofluid samples are frequently found at crime scenes; however, existing gold standard methods such as LC–MS frequently require destructive extraction of the sample before a time‐consuming analysis which puts strain on forensic analysis providers and can preclude further sample analysis. This study presents the application of sheath‐flow probe electrospray ionization‐mass spectrometry (sfPESI–MS) to the direct analysis of drug metabolites in dried blood spots (DBS) as a high throughput, minimally destructive alternative. Methods A rapid direct analysis method using a sfPESI ionisation source coupled to an Orbitrap Exactive mass spectrometer was applied to detect cocaine metabolites (benzoylecgonine, BZE, cocaethylene, CE, and ecgonine methyl ester, EME) from DBS. An optimisation study exploring the use of different chemical modifiers (formic acid and sodium acetate) in the sfPESI probe extraction solvent was conducted to enhance the sensitivity and reproducibility of the sfPESI–MS method. Results Optimisation of the extraction solvent significantly enhanced the sensitivity and reproducibility of the sfPESI–MS method. A quantitative response over a five‐point calibration range 0.5 to 10 μg/ml was obtained for BZE (R2 = 0.9979) and CE (R2 = 0.9948). Limits of detection (LOD) of 1.31, 0.29 and 0.15 μg/ml were achieved for EME, BZE and CE, respectively, from 48 h aged DBSs with % RSD (relative standard deviation) across the calibration range ranging between 19%–28% for [BZE + H]+, 13%–21% for [CE + H]+ and 12%–29% for [EME + H]+. Conclusions A rapid (< 20 s) quantitative method for the direct analysis of cocaine metabolites from DBS which requires no prior sample preparation was developed. Although the LOD achieved for BZE (LOD: 0.29 μg/ml) was above the UK threshold limit of exposure for drug driving (0.05 μg/ml), the method may be suitable for use in identifying overdose in forensic analysis.


| INTRODUCTION
Biological samples such as dried blood spots (DBSs) can provide useful information in both criminal investigations and identifying accidental or deliberate exposure to chemicals. The presence of drugs and/or their metabolites in a sample can give additional information about a suspect, perpetrator or victim in a criminal investigation or be used to identify the chemical agent responsible for intoxication or poisoning. In a forensic setting though several different bodily fluids are commonly found in crime scene investigations, blood is the most widely studied forensic biofluid; however, other fluids such as saliva, semen, urine, sweat and vaginal fluid have also played a vital role in specific investigations. 1 The identification of a biofluid at a crime scene usually will begin with a presumptive test, which is typically done on site to determine the identity of the stain, before sending the sample to a specialist laboratory for confirmatory analysis, including DNA profiling, to potentially identify the suspect. 1,2 Presumptive tests are typically simple colorimetric tests that indicate the presence of a particular fluid by a colour change which is induced by the addition of a reagent (e.g. luminol solution reacting with iron in blood), 3 with each body fluid requiring a specific presumptive test to enable identification. 1 There are limitations which apply to presumptive tests; some may lack specificity and react with multiple biofluids or other chemical interferents which may be present at the scene causing false-positive results. 4 In addition, many common presumptive tests are destructive to the sample and may render it useless for subsequent confirmatory tests and/or DNA analysis. 1 This is a particular issue when small volumes of the sample are present. 5 In recent years, analytical methods have been developed to address this issue, and a range of different spectroscopic methods have been applied to identify body fluids. These methods include Raman spectroscopy [6][7][8] and Fourier transform-infrared spectroscopy (FT-IR) 8,9 both of which are non-destructive methods that will not damage the sample in the way a conventional presumptive test may.
Mass spectrometry (MS) is a particularly attractive technique for biofluid identification and screening as it possesses a high degree of sensitivity and low limits of detection, and can identify lowabundance analytes in a biofluid sample. 10,11 This gives a mass spectrometric method the ability to gain additional information about the biofluid sample and the individual it originated from, in addition to identifying what the biofluid is. For example, the presence of pharmaceuticals, narcotics or poisons in the biofluid sample can be determined, which may be beneficial to the investigation in giving further insight into the crime being committed and reduce the load on testing laboratories. 12 In the confirmatory testing role, MS has long been a gold-standard method for analysing illicit substances in body fluids. However, the majority of standard methods and well-used approaches such as liquid chromatography-mass spectrometry (LC-MS) require destructive extraction, modification and preconcentration techniques to be employed before analysis. 1,13 Ambient ionization MS methods are capable of analysing samples in-situ with minimal sample preparation and have been used both to identify biofluids 14,15 and to detect specific targeted compounds present in biofluids, including drug compounds 16,17 and disease biomarkers. 17,18 Probe electrospray ionization (PESI), a method developed by Hiraoka et al at the University of Yamanishi in Japan, 19 is a particularly valuable method for forensic biofluid analysis as it requires no sample preparation and has short analysis times. In addition, PESI has been shown to consume only a few picolitres of sample during analysis which makes it useful in forensic scenarios where only trace levels of sample may be present. 20 PESI has been applied to analyse a range of analytes, including the detection of illicit drugs in body fluids. 21 In such cases the PESI method was shown to be able to detect cocaine with a limit of detection (LOD) of 0.05 μg/ml in 1:1 diluted urine, 0.02 μg/ml in 1:1 diluted oral fluid and 0.2 μg/ml in plasma after a protein precipitation step, with good quantitative performance (% RSDs [relative standard deviation] of 9.55% in urine, 10.44% in oral fluid and 14.81% in plasma). 21 A limitation of the PESI method is that it is not capable of analysing solid samples, which restricts its utility towards samples such as DBS.
A subsequent development of the PESI technique, sheath-flow PESI (sfPESI) enabled the direct analysis of solid samples. 22,23 A sfPESI interface uses a fine needle contained within a solvent-filled gel-loading tip, with the needle protruding slightly (c. 0.1 mm) from the base of the gel-loading tip. [23][24][25] Analytes are then extracted from a surface by touching the sfPESI probe to the sample surface, which can be either a wet or dried material, for 5 s in which liquid extraction from the surface occurs. 25 A high voltage (≤ 2.5 kV) is applied to facilitate an electrospray through the probe for a short duration, c. 5 s. [22][23][24][25][26] There are a number of advantages in this method. Due to the small diameter of the needle of the sfPESI probe, only about 1 mm 2 of the sample surface ( Figure S1b [supporting information]) is affected by the extraction making it minimally destructive. Most notably probe electrospray methods, including sfPESI, exhibit a sequential ionization mechanism where analytes and matrix compounds show a degree of separation based on their surface activity. 23,27 This study showed that when a cytochrome C protein sample was mixed with a 100-fold excess of a non-ionic detergent (Triton X100) and analysed using PESI. 27 The highly surface active detergent was emitted in the first 10 s of the analysis, whereas the less surface active protein was emitted later between 30 and 50 s, enabling improved detection of the protein as it reduced ion suppression by the Triton X100. 27 When the same sample was analysed using nano-electrospray, severe ion suppression of the cytochrome C caused by the presence of the detergent was noted. 27 The same study also used PESI to analyse the peptide hormone insulin in the presence of an excess of NaCl. In this example, the peptide was eluted first with the Na + ions only being electrosprayed at the final stage of the electrospray implying enrichment of NaCl in the main droplet on the PESI emitter over time. NaCl clusters were observed only in the final stages of the analysis which showed an almost-complete separation of the peptide from the salt. These data confirm that the ionization of solvated analytes in PESI occurs in the order of decreasing surface activity. 27 sfPESI-MS has previously been used to identify biofluid samples using a profiling approach which was able to distinguish blood, urine, saliva and semen in both fresh and dried samples, demonstrating the ability of sfPESI to extract small metabolites like creatinine, carnitine and urea as well as larger phospholipids. 25,28 That study also investigated sample ageing, and it was noted that responses associated with certain phospholipid ions decreased as the DBS aged. 25 Although demonstrating an ability to profile biofluids, however, this method has not yet been applied to perform quantitative measurements of compounds such as drug metabolites in dried samples.
In this study, sfPESI-MS is applied to analyse cocaine metabolites  Whole human blood samples were taken by venous draw, from a single consenting volunteer using a protocol approved by the ethical committee of Loughborough University (Ethics statement number R18-P034). The blood samples were aliquoted out and then spiked with each cocaine metabolite standard into a consistent amount of whole blood before spotting on to glass microscope slides (25.4 Â 76.2 Â 1.2 mm, Sail Brand, China). The blood spot specimens were stored in a dark cupboard at room temperature (22 ± 1 C) for 48 h to enable them to dry prior to sfPESI-MS analysis.

| sfPESI emitter construction and interfacing
The sfPESI source ( Figure 1) was constructed from a gel-loading pipette tip (0.5-20 μl GELoader tip, Eppendorf AG, Hamburg, Germany) and a fine stainless steel acupuncture needle (J type OD 0.12 mm Â L 30 mm, Seirin, Shizuoka, Japan) which was inserted into the gel-loading tip ( Figure 1A). The gel-loading tip was filled with c. Initial sfPESI-MS data for the extraction solvent efficiency study were acquired using the interface configuration shown in Figure 1B, which has been reported elsewhere. 25

| sfPESI-MS analysis method
Initial sfPESI-MS experiments were performed using a 50%v/v ethanol/water solution as the extraction solvent; however, after an optimisation study, a 50%v/v ethanol/water solution containing 0.5mM sodium acetate (SA) and 0.1% formic acid (FA) was used as an optimised extraction solvent for the sfPESI emitter.
For the optimisation study, liquid sample solutions of 5 μg/ml BZE were analysed by dipping the emitter into the solution. For DBS analysis, samples were extracted from DBS by gently touching the sfPESI emitter onto the sample surface for 5 s, which allowed the extraction of analytes from the surface to occur by capillary phenomena, with an area of < 1 mm 2 being sampled ( Figure S1 [supporting information]). In addition, for quantitative experiments, a background blank signal was measured by extracting directly from the glass slide surface before commencing DBS analysis, and additional background blank samples were analysed between DBS analyses to ensure no carryover occurred between each replicate DBS analysis.
A Thermo Exactive Orbitrap mass spectrometer (ThermoFisher Scientific, Bremen, Germany) was used for all sfPESI-MS analyses, and the optimised settings of the mass spectrometer are shown in Table S1 (supporting information). The sfPESI emitter was vertically

| Data analysis
Data were collected and analysed using Thermo Xcalibur software (Version 3.0, ThermoFisher Scientific, Bremen, Germany) with a < 5 ppm mass error (or mass tolerance). Mass spectral intensities were obtained after subtraction of the background mass spectrum from the glass slide from the DBS mass spectrum to remove background ions which originate from either the glass slide surface or the extraction solvent. From the mass spectral data acquired, fundamental statistics, such as the mean and relative standard deviation (% RSD) of mass spectral intensities obtained, were used to compare the results. 29 Outliers were rejected using Dixon's Q-test when assessing the reliability of analysis results, and a minimum of six replicates were measured for each analytical sample to reduce random error and improve the precision of data obtained. 29 Limits of detection (LOD = 3σ/m) were calculated by taking thrice the calculated standard deviation (σ) in the sample response at the lowest sample concentration and dividing by the slope value (m) of the linear correlation graphs generated. 29 All graphs were created and analysed through the Origin software (Version 2019, OriginLab Corporation, USA).

| RESULTS AND DISCUSSION
sfPESI-MS was applied to analyse cocaine metabolites (BZE, CE and EME) directly from DBSs as a rapid, direct and minimally destructive analysis method with no requirement for prior sample preparation.
A list of the cocaine metabolites under investigation with their molecular structures and expected adduction forms in sfPESI-MS analysis are shown in Table 1. Prior to commencing analysis of cocaine metabolites from DBS, an optimisation study was performed using liquid standard samples to improve the sensitivity and reproducibility of the sfPESI-MS method.

| Extraction solvent chemical modifier optimisation
An aqueous 50%v/v ethanol/water solution was initially used as an extraction solvent. About 5 μg/ml of a BZE standard solution in methanol was analysed as a liquid sample to avoid any variability caused by sample inhomogeneity from drying onto a surface. Ten Three intense peaks which were attributed to BZE were detected in the mass spectra obtained under these conditions ( Table 2). The  The use of chemical modifiers in the sfPESI probe extraction solvent will influence its properties when extracting multifarious species from various sample surfaces, and the extraction efficiency will depend on analyte characteristics and molecular structure. Due to the improved reproducibility of the results obtained using the combination of acidic and alkali metal modifiers compared to using an acidic modifier alone, this extraction solvent was selected to be used for quantitative DBS analysis.   32 This effect will lead to the formation of Na + -enriched droplets during the electrospray process. 32 Na + and K + ions will be extracted from the DBS by the sfPESI probe extraction solvent and will be a source of variability between replicate analyses. The addition of excess Na + ions into the extraction solvent through the addition of SA minimises the contribution from Na + and K + ions present in the DBS, thus reducing the variability observed in the replicate analyses when modifiers were used. 32 These data show the benefit of using chemical modifiers in mobile phase when performing direct analysis from DBSs using sfPESI and demonstrate the potential for quantitative analysis.

| Surface activity separation
A common issue that can arise when performing direct analysis on complex biofluids using ESI, and particularly when using mixed modifiers, is that the presence of background ions and undesired adducts can act as matrix interferences. 33,34 These interferences can mask analyte signals through the formation of matrix ion clusters 33 and reduce the overall abundance of analytes through ion suppression. 34 Using the solvent modifiers (FA and SA) shown earlier, sodium formate clusters will be readily formed through electrospray ion formation process when sodium formate is formed by nebulizing formate with sodium. 35 Sodium formate cluster ions, [NaOOCH] n Na +  Figure 2 shows that in the mass spectrum obtained from the first scan (S1), which appears < 1 s after applying the spray voltage of 2.5 kV, the cocaine metabolite ions are the dominant species without sodium formate cluster interferences being observed and with minimal interferences from background ions ( Figure 2B). However, at the second time point (the 12th scan, S12 at about 5 s) the spectrum is dominated by background ions and the sodium formate clusters are clearly observed ( Figure 2C).
This rapid separation mechanism is a known advantageous feature of PESI methods, and it was firstly described by Mandal Table S5 (supporting information). Cocaine metabolites were not detected in any of the replicate blank measurements or in the blanks performed between sample analyses. is that the LODs achieved for all three metabolites were too high to detect habitual users at the blood limit for drug driving set by the UK Department for Transport (UKDfT). 36 The UKDfT regulations classify BZE as a metabolite of an illicit drug and thus adopt a zero-tolerance approach by setting a limit of 0.05 μg/ml in blood as determined by a gas chromatography-mass spectrometry (GC-MS) assay, which is the highest amount of BZE in the blood that is thought to result from accidental exposure. 36 This is below the LOD of the instrumental configuration presented here.
The The quantitative performance of the sfPESI-MS method was evaluated for the three metabolites and showed adequate reproducibility (% RSD of ≤ 29%) and LODs which could be useful for the detection of overdose and/or severe intoxication in blood spots found at crime scenes. However, further development and interfacing with tandem mass spectrometry (MS/MS) instrumentation is required for the method to be able to detect users at the limit for accidental exposure.